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Biocontrol of Salmonella in the food industry using phage therapy Miquel Sánchez Osuna - Universitat Autònoma de Barcelona - Grau de Microbiologia BACKGROUND Food is an essential requirement of everyday life, but occasionally, contamination with pathogenic bacteria can result in illness and even death. According to the European Food Safety Authority (EFSA), food-borne illnesses are one of the leading causes of morbidity and mortality in the world, and it was estimated that approximately one third of the population may be affected by such diseases in developed countries every year. Ensuring food safety involves a wide range of microbial control measures applied to relevant steps in the food chain. Currently, the most effective means to limit microbial growth are good hygiene in the production and proper use of biocides and disinfectants. However, these treatments are inefficient and can produce organoleptic variations or the possibility of leaving toxic waste. Consequently, the development of effective and safe natural methods has been an interesting topic during the last years. Phage therapy emerges as one of the solutions becoming a good approach to reduce the incidence of food-borne diseases. BIOCONTROL OF Salmonella In the European Union, salmonellosis is the second reported zoonotic disease in humans. Although the numbers of reported outbreaks caused by vegetables and products has increased, poultry and derivates are still the common sources of Salmonella. The following table shows several studies concerning phage therapy against Salmonella. Biocontrol of Salmonella in primary production Year Product Phage(s) Strategy Conclusions 2007 Poultry φ151, φ25, φ10 Oral delivery Reduction of CFU with phages φ151 and φ10, not observed with φ25. Significant numbers of phages are required to adsorb to individual host cells. Importance of accessible receptors on cell surface to allow adsorption. 2007 Poultry CB4φ, WT45φ (cocktail) Oral delivery Significant reduction at 24h, not at 48h. Emergence of resistant cells. 2012 Poultry UAB_Phi20, UAB_Phi78, UAB_Phi87(cocktail) Oral delivery Importance of administering the phage prior to infection and continued administration to achieve significant protection. 2012 Poultry φCJ07 Oral delivery (via feed) CFU reduction in uninfected chickens. ΦCJ07 to prevent cross contamination in poultry. 2013 Poultry φst1 Intracloacal delivery Salmonella was not detected at and after 24h. Intracloacal administration avoids contact with stomach. 2013 Poultry (eggs) F1O55S, F12013S (cocktail) Spray delivery F1O55S and F12013S reduce Salmonella horizontal transmission during egg incubation. 2011 Swine Phage cocktail Oral delivery Reduction of Salmonella within 96h. Bacteriophages can reduce Salmonella in swine. Biocontrol of Salmonella in postharvest food Year Product Phage(s) Strategy Conclusions 2003 Chicken skin P22, 29C Applied on top MOI (multiplicity of infection) 100-1000 caused more reduction than MOI 1 and eradicated resistant strains. 2001 Fresh-cut fruit Phage cocktail Added to foods Significant CFU reduction on melon but not on apple. pH of apples inactive bacteriophages. 2004 Sprouting seeds Phage cocktail Applied by immersion Significant reduction. It is important to develop phage cocktails to control a wide range of contaminants. 2012 Ready-to-eat food F01-E2 Added to foods Significant reduction of CFU in hot dogs, cooked sliced turkey breast, mixed seafood and chocolate milk at 15ºC. FACTORS AFFECTING THE EFFECTIVENESS OF PHAGE THERAPY The effectiveness of phage applications against pathogenic bacteria depends on several factors. CONCLUDING REMARKS AND FUTURE PERSPECTIVES - This work reflects that bacteriophages are a remarkable alternative to control and eradicate pathogenic bacteria in primary production and postharvest food. - The application of phage therapy is strengthened by the number of companies around the world investing in the production of phage-based products for use in the food industry. SALMONELEX™, for example, is a phage-based product to control Salmonella in the food chain. - However, regulatory agencies are careful with the application of bacteriophages because of the lack of scientific evidence through clinical trials fully supervised by ethics committees and regulatory compliance standards. PHAGE THERAPY Historically, phage therapy arose to control resistant bacteria because of the widespread problem of antibiotic resistance coupled with the paucity of new antibacterial drugs. Even so, the interest has been renewed for the control of bacteria in other areas, including food. BACTERIOPHAGES IN FOOD INDUSTRY The use of bacteriophages to promote food safety can be mainly done at three different stages along the food chain. To reduce pathogen colonization in animals during primary production. Disinfection of food contact surfaces and equipments. Directly in postharvest food, such as meat, fresh fruit, vegetables and processed RTE foods. Directly in postharvest food, such as meat carcasses, fresh fruit, vegetables and processed ready-to-eat (RTE) foods. Advantages and disadvantages of phage therapy Advantages Disadvantages Highly specific, rapid bacterial killing, ability to self-replicate and natural. Narrow host range. Mixture (cocktail) usually required. Minimal disruption to regular microbiota. Knowledge of biology often required. Does not affect organolpetic properties. Negative consumer perception. Abundant in natural environments. Effective in biofilms. REFERENCES Endersen L., O’Mahony J., Hill C., Ross R. P., McAuliffe O., Coffey A. 2014. Phage therapy in the food industry. Annual Review of Food Science and Technology. 5: 327-349. // European Food Safety Authority, European Centre for Disease Prevention and Control. 2011. The European Union summary report on trends and sources of mzoonoses, zoonotic agents and food-borne outbreaks in 2009. The European Food Safety Authority Journal.9:2090. // Ly-Chatain, M. H. 2014. The factors affecting effectiveness of treatment in phages therapy. Front Microbiol. 5: 1-7. // Sillankorva S. M., Oliveira H., Azeredo J. 2012. Bacteriophages and their role in food safety. Int J Microbiol. Volume 2012: 1-13. Dose and moment of treatment. Early treatment (or prior to infection), continued administration Concentration. MOI from 0.01 to 100. Neutralization by immune system or other compounds. Repeating the administration, increasing the doses, encapsulation. Administration. Oral, intracloacal, topical, intraperitoneal, intranasal or intravenous. Food conditions (pH, temperature…) Encapsulation. Accessibility to target bacteria (intracellular cells are inaccessible, limited diffusion in solid matrices). Specificity and resistance to phage. Use of a cocktail.
